Premenopausal women are relatively protected against clinically apparent coronary heart disease (CHD), compared to age-matched males. Although the difference has been accepted to be hormonally-mediated, this concept has been challenged by results of prospective, large clinical trials and cohort studies. This has intensified the interest in a finer dissection of both the molecular and physiologic aspects of estrogen's effects on the vasculature. CHD is the result of a dynamic process that includes modulation of vasoreactivity, vascular inflammation and injury, cell growth and matrix deposition, all processes affected by endothelium-derived nitric oxide (NO). In addition to its important effects on gene expression, estrogen, through engagement of membrane receptors, stimulates rapid endothelial cell (EC) signaling. We have demonstrated that membrane-impermeant 172-estradiol (E2) activates endothelial NO synthase (eNOS) in a c-Src/phosphatidylinositol-3 kinase/Akt-dependent fashion, and that this is most efficiently mediated by an EC membrane-localized ER1 isoform, ER46. Our hypothesis is that estrogen-stimulated eNOS activation is mediated through an ER46- centered, membrane signalsome in ECs, and that these signaling responses are dependent on both specific and direct interactions between ER46, c-Src and scaffold molecules. Furthermore, we hypothesize that ER46 has a novel membrane topology, conferred through a conserved hydrophobic sequence and membrane insertion characteristics. Finally, we believe that the rapid endothelial activation induced by these signaling events is a critical feature of a relatively protected vascular environment. Specific proposals now include: (1) to define molecular domains of ER46, c-Src and the modulator of nongenomic activity of ER (MNAR) required for direct interaction, membrane localization and E2-triggered, rapid responses by (a) co-immunoprecipitation, (b) fluorescence resonance energy transfer (FRET), (c) membrane impermeant estrogen binding, and (d) eNOS activation responses;(2) to further define ER46 membrane targeting, topology and function by (a) subcellular localization and functional studies with ER46 mutated in putative transmembrane spanning and lipid modification domains, and (b) purifying recombinant ER46 and generating 2-dimensional crystals, in preparation of in-membrane cyro-EM structural analysis;and (3) to demonstrate the functional significance of alternative ER1 isoforms in NO-dependent vascular responses in/ex vivo, using established models of (a) aortic ring tension, and (b) ear microvessel flow/permeability, in the ER46-retained ER1-neo/2 knockout mouse. These studies will provide novel molecular and structural information regarding an efficiently membrane-targeted ER1 isoform in the endothelium, and lay the foundation for targeted pharmacotherapy in cardiovascular disease, of great relevance in women's health. PUBLIC HEALTH RELEVANCE: Premenopausal women are relatively protected from coronary heart disease, compared to males of the same age. This work seeks to define the ways in which the hormone estrogen maintains healthy blood vessel cells, with an eye toward eventually targeting blood vessels with modulators that protect blood vessels, both in women and men.